The focus of this proposal is to develop a biomaterial platform that will enable the 3-D culture of valvular interstitial cells (VICs) and subsequent manipulation of the cellular microenvironment to promote or suppress selected cell functions. Through this type of three-dimensional culture system, we believe that scaffolds will be identified that will facilitate the regeneration of functional valve leaflets. Specifically, we propose to determine the effect of specific matrix interactions (e.g., fibronectin, heparin) and soluble cytokines (e.g., bFGF, TGF-[unreadable]1) on VIC function in 2D (aim 1). These results will then be used to develop highly regulated biomaterials niches to control VIC function and matrix production in 3D (aims 2 &3). We plan to design 3-D scaffold chemistries that will permit VIC viability and proliferation, as well as promote expression and activation of VICs to a myofibroblast phenotype, which is prevalent during valve remodeling and development (aim 2). Subsequently, we will manipulate the degradation- dependent scaffold properties to support extracellular matrix deposition and functional tissue regeneration (aim 3). The experimental approach for aims 2 and 3 will be to photoencapsulate VICs in poly (ethylene glycol) (PEG) and hyaluronan (HA)-based copolymer hydrogels that will be systematically modified with matrix components to support VIC interactions. In addition, bFGF and TGF-[unreadable]1 will be introduced into the cell-gel constructs through bulk and localized delivery methods. Confocal microscopy will be used to directly visualize cell viability over time. BRDU incorporation and gene expression with time, as determined by real time RT-PCR, immunostaining, and in situ hybridization will be used to assess VIC proliferation and myofibroblast differentiation. Functional activity of VICs will be assessed by measuring cell- cell communication, extracellular matrix secretion, and evolving mechanical properties. The effects of gel chemistry, especially the introduction of matrix components and cyotokines, on VIC function will be screened by culturing these cell-laden hydrogels in vitro. Results from these aims will be used to identify hydrogel formulations that permit VIC function, promote controlled myofibroblast differentiation, and facilitate matrix formation. These in vitro results will then provide the foundation to select formulations for future development of functional valve structures in appropriately designed bioreactors. This proposal aims to prepare biomaterial microenvironments that incorporate signals to actively promote the function of heart valve cells for tissue regeneration and to improve the field's understanding of heart valve function by providing a more biomimetic 3D culture system for heart valve cells. If successful, this strategy will prolong the duration and function of tissue-based valve replacements, especially for children.